Somatic embryogenesis is one of a number of methods known in the art for the propagation of desireable species or varieties of plants. There are many advantages, however, which favor somatic embryogenesis as a propagative method of choice. One advantage is that a plant which has a known and desirable phenotype can be chosen as the source of cells, and, in accordance with somatic embryogenesis techniques, these cells can be rapidly cultured into many genetically uniform embryos. The resulting embryos can then be cultivated into entire plants possessing roots and shoots. Thus, in accordance with this technique, plants with the same desirable phenotype as the parent can be mass produced, potentially at costs comparable to and often more quickly and with better genetic uniformity than other propagative techniques such as, for example, the generation of field grown seed.
The process of producing embryos from somatic tissues, however, requires the development of an embryogenic culturing medium and protocol suitable for the particular type of tissue. Various media and procedures have been attempted, but only some have been met with success.
Stuart et al., U.S. Pat. No. 4,801,545, for example, describes a medium for culturing somatic plant cells comprising 7.5 to 300 mM maltose along With a balanced salt solution containing plant cell nutritional and growth requirements. Also disclosed and claimed is a method of producing embryonic tissue from somatic tissue, specifically leguminosae somatic tissue, wherein the somatic tissue is regenerated to form embryonic tissue in a growth medium containing 7.5 to 300 mM maltose and at least one amino acid selected from the group consisting of proline, alanine and glutamine in an amount sufficient to stimulate somatic embryogenesis or embryo conversion.
Promoting successful somatic embryogenesis with woody species of plants, such as the gymnosperms, has been much more difficult than with foliage plants. However, researchers have achieved some success in this area also.
Abo El-Nil, U.S. Pat. No. 4,217,730, for example, describes methods for generating embryoids and plantlets from gymnosperm plant tissue through the use of tissue culture techniques. In one embodiment, the method comprises culturing excised gymnosperm plant tissue on a medium containing mineral nutrients, organic nutrients and auxins (as the sole exogenous plant hormone) until a soft callus tissue has developed, transferring the callus tissue into a suspension medium containing similar nutrients and hormone components (again, wherein auxins are the sole exogenous plant hormone), and agitating the suspension gently to produce embryoids. In another embodiment, the disclosed method comprises culturing excised gymnosperm plant tissue on a first medium containing mineral salts, organic nutrients, and an auxin selected from the group consisting of 2-4-dichlorophenoxyacetic acid and naphthalene-2-acetic acid until the formation of a soft callus, transferring the callus tissue into a suspension medium in which the callus is gently agitated, the suspension medium being similar in nutrient and hormone components to the first medium, and removing the resultant embryoids and placing them on a solid growth medium. MS basal medium, employed therein, includes various inorganic salts, as well as organic compounds, such as 3-indoleacetic acid, kinetin and sucrose, as set forth in Murashige and Skoog, Physiol. Plant. Vol. 115, pp. 473-497, at 475 (1962) (cited therein).
Gupta and Durzan, Biotechnology, Vol. 5, pp. 147-151 (1987), describes the somatic polyembryogenesis of the loblolly pine (Pinus taeda L.) using multiple phase culturing methodology employing MS basal medium variously supplemented with such compounds as 2,4-dichlorophenoxyacetic acid, kinetin, benzyladenine and sucrose. MS basal medium, as contemplated therein, includes various inorganic salts, as well as organic compounds, such as 3-indoleacetic acid, kinetin and sucrose, as noted in Gupta and Durzan, Biotechnology, Vol. 5, pp. 147-151, at 150-151 (1987), and Murashige and Skoog, Physiol. Plant. Vol. 115, pp. 473-497, at 475 (1962) (cited therein).
Durzan and Gupta, Plant Sciences. Vol. 52, pp. 229-235 (1987), describes the somatic embryogenesis and polyembryogenesis in Douglas-fir (Pseudotsuga menzesii) cell suspension cultures using multi-step culturing techniques employing MS basal medium and DCR basal medium variously supplemented with such compounds as 2-4-dichlorophenoxyacetic acid, kinetin, N-benzylaminopurine, abscisic acid and sucrose. DCR basal medium, as contemplated therein, includes various inorganic salts, as well as organic compounds such as sucrose, as set forth in Gupta and Durzan, Plant Cell Rep., Vol. 4, pp. 177-179, at 177 (1985) (cited therein).
Mehra-Palta, U.S. Pat. No. 4,417,417, discloses methods of clonal propagation of gymnosperms in which excised gymnosperms tissue is treated on a nutrient medium containing cytokinin for a time sufficient to induce formation of adventitious buds. If desired, an auxin, such as indole-3-butyric acid, may be included in this step. The bud containing tissue is then transferred to a nutrient medium free of exogenous growth factors and maintained thereon until the induced adventitious buds produce rootable shoots. The rootable shoots may then be rooted using conventional techniques, but are preferably treated on a nutrient medium containing an auxin, such as indole-3-butyric acid, for a time sufficient to induce formation of adventitious roots, and are then transferred to a nutrient medium free of exogenous growth factors until the shoots are rooted. Suitable nutrient medium includes various inorganic salts, as well as organic compounds, such as sucrose.
Despite some success in developing suitable culture media and culturing processes for somatic embryogenesis, new and/or better media and methods, particularly for the commercially significant gymnosperm forest trees, are needed. The present invention is directed to this important end.